Kevin E. Conley

Oxidative capacity and ageing in human muscle

1 This study determined the decline in oxidative capacity per volume of human vastus lateralis muscle between nine adult (mean age 38.8 years) and 40 elderly (mean age 68.8 years) human subjects (age range 25‐80 years). We based our oxidative capacity estimates on the kinetics of changes in creatine phosphate content ([PCr]) during recovery from exercise as measured by 31P magnetic resonance (MR) spectroscopy. A matched muscle biopsy sample permitted determination of mitochondrial volume density and the contribution of the loss of mitochondrial content to the decline in oxidative capacity with age. 2 The maximal oxidative phosphorylation rate or oxidative capacity was estimated from the PCr recovery rate constant (kPCr) and the [PCr] in accordance with a simple electrical circuit model of mitochondrial respiratory control. Oxidative capacity was 50 % lower in the elderly vs. the adult group (0.61 ± 0.04 vs. 1.16 ± 0.147 mM ATP s−1). 3 Mitochondrial volume density was significantly lower in elderly compared with adult muscle (2.9 ± 0.15 vs. 3.6 ± 0.11 %). In addition, the oxidative capacity per mitochondrial volume (0.22 ± 0.042 vs. 0.32 ± 0.015 mM ATP (s %)−1) was reduced in elderly vs. adult subjects. 4 This study showed that elderly subjects had nearly 50 % lower oxidative capacity per volume of muscle than adult subjects. The cellular basis of this drop was a reduction in mitochondrial content, as well as a lower oxidative capacity of the mitochondria with age.

Separate measures of ATP utilization and recovery in human skeletal muscle.

1. The chemical changes during contractile activity were separated from recovery metabolism in the forearm flexor musculature in normal human subjects using 31P nuclear magnetic resonance (NMR) spectroscopy. Percutaneous, supramaximal twitch stimulation of the median and ulnar nerves was used in combination with temporary ischaemia of the forearm to characterize the summed ATPase activity. The recovery following restoration of blood flow provided a measure of oxidative ATP synthesis activity. These processes were measured based on the dynamics of creatine phosphate (PCr) content. 2. Muscle oxygen stores were depleted using ischaemia without stimulation as indicated by PCr breakdown after 250 +/‐ 33 s (mean +/‐ S.D.; n = 5), which provided a measure of the basal metabolic rate (0.008 +/‐ 0.002 mM s‐1, n = 5). 3. The PCr breakdown rate during twitch stimulation of the oxygen‐depleted muscle was constant at 1 Hz at 0.15 +/‐ 0.03 mM PCr per second or per twitch (n = 8). A constant cost per twitch was found from 0.5 to 2 Hz stimulation (depletion of PCr per twitch = 0.15 mM per twitch). 4. No net anaerobic recovery of PCr was found during a 2 min post‐stimulation ischaemia. 5. Upon restoration of blood flow, PCr recovery followed an exponential time course with a time constant of 63 +/‐ 14 s (n = 8). From these recovery rates, the capacity for oxidative phosphorylation was estimated to be 0.4 mM s‐1. 6. This experimental approach defines a non‐invasive and quantitative measure of human muscle ATPase rate and ATP synthetase rate.

Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism

In response to growth factors, mTOR (mammalian target of rapamycin) has been identified as a central component of the signalling pathways that control the translational machinery and cell growth. Signalling through mTOR has also been shown to be necessary for the mechanical load-induced growth of cardiac and skeletal muscles. Although the mechanisms involved for mechanically induced activation of mTOR are not known, it has been suggested that activation of PI3K (phosphoinositide 3-kinase) and protein kinase B (Akt), via the release of locally acting growth factors, underlies this process. In the present study, we show that mechanically stimulating (passive stretch) the skeletal muscle ex vivo results in the activation of mTOR-dependent signalling events. The activation of mTOR-dependent signalling events was necessary for an increase in translational efficiency, demonstrating the physiological significance of this pathway. Using pharmacological inhibitors, we show that activation of mTOR-dependent signalling occurs through a PI3K-independent pathway. Consistent with these results, mechanically induced signalling through mTOR was not disrupted in muscles from Akt1-/- mice. In addition, ex vivo co-incubation experiments, along with in vitro conditioned-media experiments, demonstrate that a mechanically induced release of locally acting autocrine/paracrine growth factors was not sufficient for the activation of the mTOR pathway. Taken together, our results demonstrate that mechanical stimuli can activate the mTOR pathway independent of PI3K/Akt1 and locally acting growth factors. Thus mechanical stimuli and growth factors provide distinct inputs through which mTOR co-ordinates an increase in the translational efficiency.

Decline in isokinetic force with age: muscle cross-sectional area and specific force

Abstract Humans produce less muscle force (F) as they age. However, the relationship between decreased force and muscle cross-sectional area (CSA) in older humans is not well documented. We examined changes in F and CSA to determine the relative contributions of muscle atrophy and specific force (F/CSA) to declining force production in aging humans. The proportions of myosin heavy chain (MHC) isoforms were characterized to assess whether this was related to changes in specific force with age. We measured the peak force of isokinetic knee extension in 57 males and females aged 23–80 years, and used magnetic resonance imaging to determine the contractile area of the quadriceps muscle. Analysis of MHC isoforms taken from biopsies of the vastus lateralis muscle showed no relation to specific force. F, CSA, and F/CSA decreased with age. Smaller CSA accounted for only about half of the 39% drop in force that occurred between ages 65–80 years. Specific force dropped about 1.5% per year in this age range, for a total decrease of 21%. Thus, quantitative changes in muscle (atrophy) are not sufficient to explain the strength loss associated with aging.

Skeletal muscle NAMPT is induced by exercise in humans

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is responsible for the first and rate-limiting step in the conversion of nicotinamide to nicotinamide adenine dinucleotide (NAD+). NAD+ is an obligate cosubstrate for mammalian sirtuin-1 (SIRT1), a deacetylase that activates peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), which in turn can activate mitochondrial biogenesis. Given that mitochondrial biogenesis is activated by exercise, we hypothesized that exercise would increase NAMPT expression, as a potential mechanism leading to increased mitochondrial content in muscle. A cross-sectional analysis of human subjects showed that athletes had about a twofold higher skeletal muscle NAMPT protein expression compared with sedentary obese, nonobese, and type 2 diabetic subjects (P < 0.05). NAMPT protein correlated with mitochondrial content as estimated by complex III protein content (R(2) = 0.28, P < 0.01), MRS-measured maximal ATP synthesis (R(2) = 0.37, P = 0.002), and Vo(2max) (R(2) = 0.63, P < 0.0001). In an exercise intervention study, NAMPT protein increased by 127% in sedentary nonobese subjects after 3 wk of exercise training (P < 0.01). Treatment of primary human myotubes with forskolin, a cAMP signaling pathway activator, resulted in an approximately 2.5-fold increase in NAMPT protein expression, whereas treatment with ionomycin had no effect. Activation of AMPK via AICAR resulted in an approximately 3.4-fold increase in NAMPT mRNA (P < 0.05) as well as modest increases in NAMPT protein (P < 0.05) and mitochondrial content (P < 0.05). These results demonstrate that exercise increases skeletal muscle NAMPT expression and that NAMPT correlates with mitochondrial content. Further studies are necessary to elucidate the pathways regulating NAMPT as well as its downstream effects.

Skeletal Muscle Mitochondrial Energetics Are Associated With Maximal Aerobic Capacity and Walking Speed in Older Adults

BACKGROUND Lower ambulatory performance with aging may be related to a reduced oxidative capacity within skeletal muscle. This study examined the associations between skeletal muscle mitochondrial capacity and efficiency with walking performance in a group of older adults. METHODS Thirty-seven older adults (mean age 78 years; 21 men and 16 women) completed an aerobic capacity (VO2 peak) test and measurement of preferred walking speed over 400 m. Maximal coupled (State 3; St3) mitochondrial respiration was determined by high-resolution respirometry in saponin-permeabilized myofibers obtained from percutanous biopsies of vastus lateralis (n = 22). Maximal phosphorylation capacity (ATPmax) of vastus lateralis was determined in vivo by (31)P magnetic resonance spectroscopy (n = 30). Quadriceps contractile volume was determined by magnetic resonance imaging. Mitochondrial efficiency (max ATP production/max O2 consumption) was characterized using ATPmax per St3 respiration (ATPmax/St3). RESULTS In vitro St3 respiration was significantly correlated with in vivo ATPmax (r (2) = .47, p = .004). Total oxidative capacity of the quadriceps (St3*quadriceps contractile volume) was a determinant of VO2 peak (r (2) = .33, p = .006). ATPmax (r (2) = .158, p = .03) and VO2 peak (r (2) = .475, p < .0001) were correlated with preferred walking speed. Inclusion of both ATPmax/St3 and VO2 peak in a multiple linear regression model improved the prediction of preferred walking speed (r (2) = .647, p < .0001), suggesting that mitochondrial efficiency is an important determinant for preferred walking speed. CONCLUSIONS Lower mitochondrial capacity and efficiency were both associated with slower walking speed within a group of older participants with a wide range of function. In addition to aerobic capacity, lower mitochondrial capacity and efficiency likely play roles in slowing gait speed with age.

Nuclear receptor/microRNA circuitry links muscle fiber type to energy metabolism

The mechanisms involved in the coordinate regulation of the metabolic and structural programs controlling muscle fitness and endurance are unknown. Recently, the nuclear receptor PPARβ/δ was shown to activate muscle endurance programs in transgenic mice. In contrast, muscle-specific transgenic overexpression of the related nuclear receptor, PPARα, results in reduced capacity for endurance exercise. We took advantage of the divergent actions of PPARβ/δ and PPARα to explore the downstream regulatory circuitry that orchestrates the programs linking muscle fiber type with energy metabolism. Our results indicate that, in addition to the well-established role in transcriptional control of muscle metabolic genes, PPARβ/δ and PPARα participate in programs that exert opposing actions upon the type I fiber program through a distinct muscle microRNA (miRNA) network, dependent on the actions of another nuclear receptor, estrogen-related receptor γ (ERRγ). Gain-of-function and loss-of-function strategies in mice, together with assessment of muscle biopsies from humans, demonstrated that type I muscle fiber proportion is increased via the stimulatory actions of ERRγ on the expression of miR-499 and miR-208b. This nuclear receptor/miRNA regulatory circuit shows promise for the identification of therapeutic targets aimed at maintaining muscle fitness in a variety of chronic disease states, such as obesity, skeletal myopathies, and heart failure.

Glycolysis is independent of oxygenation state in stimulated human skeletal muscle in vivo

1 We tested the hypothesis that the cytoplasmic control mechanism for glycolysis is affected by the presence of oxygen during exercise. We used a comparison of maximal twitch stimulation under ischaemic and intact circulation in human wrist flexor and ankle dorsiflexor muscles. 31P magnetic resonance spectroscopy followed the phosphocreatine (PCr), Pi and pH dynamics at 6–9 s intervals. Glycolytic PCr synthesis was determined during stimulation from pH and tissue buffer capacity, as well as the oxidative phosphorylation rate. 2 Ischaemic vs. aerobic stimulation resulted in similar glycolytic fluxes in the two muscles. The onset of glycolysis occured after fifty to seventy stimulations and the extent of glycolytic PCr synthesis was directly proportional to the number of stimulations thereafter. 3 Two‐fold differences in the putative feedback regulators of glycolysis, [Pi] and [ADP], were found between aerobic and ischaemic stimulation. The similar glycolytic fluxes in the face of these differences in metabolite levels eliminates feedback as a control mechanism in glycolysis. 4 These results demonstrate that glycolytic flux is independent of oxygenation state and metabolic feedback, but proportional to muscle activation. These results show a key role for muscle stimulation in the activation and maintenance of glycolysis. Further, this glycolytic control mechanism is independent of the feedback control mechanism that governs oxidative phosphorylation.

Reduced mitochondrial coupling in vivo alters cellular energetics in aged mouse skeletal muscle

The mitochondrial theory of ageing proposes that the accumulation of oxidative damage to mitochondria leads to mitochondrial dysfunction and tissue degeneration with age. However, no consensus has emerged regarding the effects of ageing on mitochondrial function, particularly for mitochondrial coupling (P/O). One of the main barriers to a better understanding of the effects of ageing on coupling has been the lack of in vivo approaches to measure P/O. We use optical and magnetic resonance spectroscopy to independently quantify mitochondrial ATP synthesis and O2 uptake to determine in vivo P/O. Resting ATP demand (equal to ATP synthesis) was lower in the skeletal muscle of 30‐month‐old C57Bl/6 mice compared to 7‐month‐old controls (21.9 ± 1.5 versus 13.6 ± 1.7 nmol ATP (g tissue)−1 s−1, P= 0.01). In contrast, there was no difference in the resting rates of O2 uptake between the groups (5.4 ± 0.6 versus 8.4 ± 1.6 nmol O2 (g tissue)−1 s−1). These results indicate a nearly 50% reduction in the mitochondrial P/O in the aged animals (2.05 ± 0.07 versus 1.05 ± 0.36, P= 0.02). The higher resting ADP (30.8 ± 6.8 versus 58.0 ± 9.5 μmol g−1, P= 0.05) and decreased energy charge (ATP/ADP) (274 ± 70 versus 84 ± 16, P= 0.03) in the aged mice is consistent with an impairment of oxidative ATP synthesis. Despite the reduced P/O, uncoupling protein 3 protein levels were not different in the muscles of the two groups. These results demonstrate reduced mitochondrial coupling in aged skeletal muscle that alters cellular metabolism and energetics.

Ageing, muscle properties and maximal O2 uptake rate in humans

1 This paper asks how the decline in maximal O2 uptake rate (VO2,max) with age is related to the properties of a key muscle group involved in physical activity – the quadriceps muscles. Maximal oxygen consumption on a cycle ergometer was examined in nine adult (mean age 38.8 years) and 39 elderly subjects (mean age 68.8 years) and compared with the oxidative capacity and volume of the quadriceps. 2 VO2,max declined with age between 25 and 80 years and the increment in oxygen consumption from unloaded cycling to VO2,max (ΔVO2) in the elderly was 45 % of the adult value. 3 The cross‐sectional areas of the primary muscles involved in cycling – the hamstrings, gluteus maximus and quadriceps – were all lower in the elderly group. The quadriceps volume was reduced in the elderly to 67 % of the adult value. Oxidative capacity per quadriceps volume was reduced to 53 % of the adult value. The product of oxidative capacity and muscle volume – the quadriceps oxidative capacity – was 36 % of the adult value in the elderly. 4 Quadriceps oxidative capacity was linearly correlated with ΔVO2 among the subjects with the slope indicating that the quadriceps represented 36 % of the VO2 increase during cycling. 5 The decline in quadriceps oxidative capacity with age resulted from reductions in both muscle volume and oxidative capacity per volume in the elderly and appears to be an important determinant of the age‐related reduction in ΔVO2 and VO2,max found in this study.